PROJECT SUMMARY By whole genome sequencing, we discovered highly recurring MYD88 mutations in 95-97% of Waldenstrom's macroglobulinemia (WM) patients that promote constitutive pro-survival NF-kB activation through IRAK1/IRAK4 and BTK. These findings enabled us to perform a pivotal clinical trial that led to the approval of the BTK inhibitor ibrutinib for WM by the U.S. FDA, and EMA. Despite high response rates, most responses to ibrutinib are partial, and persistent IRAK1/IRAK4 signaling appears responsible for this intrinsic resistance to ibrutinib. We therefore propose in these studies to create inducible knockdown mutants of IRAK1, IRAK4 and both IRAK1 and IRAK4 in MYD88 mutated WM cell lines to clarify the importance of IRAK1 vs. IRAK4, vs. both in mediating pro-survival signaling. We will also perform replacement experiments by transduction of kinase intact or kinase dead IRAK1 and IRAK4 to clarify the importance of scaffold versus kinase mediated pro- survival signaling. The findings from these experiments will guide development of highly selective and potent prototype inhibitors of IRAK1 (JH-X- 119-01) and IRAK1/IRAK4 (JH-I-25) that we have manufactured. Based on guidance from knockdown experiments, we will optimize the pharmacokinetic and pharmacodynamic properties of the lead inhibitor for use in human studies, and will delineate its pharmacological consequences as a single agent and in combination with ibrutinib in WM cells dependent on mutated MYD88 growth and survival signaling. Acquired resistance to ibrutinib is also an emerging problem in WM patients. We recently identified BTKCys481 mutations that abrogate ibrutinib-BTK binding in samples from half of WM patients who progressed on ibrutinib, and showed that transduction of the most common BTK mutation (BTKCys481Ser) led to activation of ERK1/2 survival signaling, inflammatory cytokine production, and ibrutinib resistance in MYD88 mutated WM cells. In recent work, we identified HCK, a SRC family member that is down-regulated at later stages of B-cell ontogeny, as an important component of mutated MYD88 survival signaling that activates BTK, as well as AKT and ERK1/2. We propose in these studies to delineate the importance of HCK blockade to overcoming acquired ibrutinib resistance mediated by mutated BTKCys481. In pursuit of this aim, we have developed highly potent and selective prototype HCK kinase inhibitors from two distinct scaffolds (SB1-G-33 and A419259) that show potent cytotoxic, HCK and BTK inhibition in BTKCys481 mutated WM cells. We propose to optimize these molecules to achieve potent target engagement, pharmacokinetic, and pharmacodynamic properties suitable for human studies, and delineate the pharmacological consequences of the lead HCK kinase inhibitor in BTKCys481 expressing ibrutinib resistant primary WM cells, and WM cell lines. We will validate the lead IRAK and HCK inhibitors developed in these studies using our in vivo WM rodent models for translation to clinical trials.